The long-term goal of this project is to determine the role of protein kinase C (PKC) and the phospholipid-hydrolyzing enzymes in the mediation of growth regulatory effects of ethanol with a special focus on fetal alcohol syndrome (FAS) and the "co-carcinogenic" effects of ethanol. The proposed work will initially determine the mechanisms by which ethanol stimulates both transient and prolonged hydrolysis of phosphatidylethanolamine (PtdEtn) by phospholipase D (PLD) and phospholipase C (PLC), respectively. Both effects require concomitant activation of PKC-epsilon (and perhaps PKC-delta by PKC activators, including phorbol 12-myristate-13-acetate (PMA), but PKC-epsilon regulates the PLD and PLC activities differently. With respect to the regulation of PLD (Aim 1), the proposed work will examine the possibility that potentiation of the stimulatory PMA effect by ethanol is due to the inhibition of p21/ras, a negative regulator of PLD. As an important tool to verify this mechanism, the effect of ethanol on the activity state of p21/ras will be determined by measuring binding of GTP to immunoprecipitated p21/ras. With respect to the regulation of PLC (Aim 2), a major task will be to determine whether PKC-epsilon potentiates the effect of ethanol per se, or PKC-epsilon stimulates the synthesis of fatty acid ethyl esters (FAEE), the possible ultimate regulator of PLC. This latter mechanism is possible because 1-chloro-2,4-dinitrobenzene (CDNB), an inhibitor of FAEE-synthase III, was found to inhibit the effect of ethanol on PLC-mediated PtdEtn hydrolysis. Various molecular species of FAEE will be synthesized to determine their possible effects on PLC activity. The work in Aim 3 will examine the role of PKC-epsilon, PKC-delta, and PLC in the mediation of inhibitory ethanol effect on nerve growth factor-induced neurite formation, a measure of differentiation, in PC12 neural cells. PKC-epsilon and PKC-delta will be overexpressed in PC12 neural cells to examine if they potentiate the inhibitory effect of ethanol on neurite formation. To probe the mediatory role of PLC, the effect of ethanol on neurite formation in control and PKC-overexpressing cells will be determined in the presence of CDNB. In the C3H/10T1/2 fibroblast system, a cellular model for carcinogenesis, ethanol potentiated the formation of transforming foci induced by a suboptimal concentration (0.25 microg/ml) of 7,12-dimethylbenz[a]anthracene. Work in Aim 4 will examine the role of PLC in the mediation of this apparent co-carcinogenic effect of ethanol (or FAEE), by using the inhibitor CDNB. The projected work will likely reveal that the effects of ethanol (or FAEE) on neural cell differentiation and carcinogenesis involve specific PKC isozymes as well as increased PtdEtn hydrolysis. This outcome could identify PtdEtn-specific PLC as a possible site of intervention to alleviate these effects of ethanol.